Method of producing dextranase

ABSTRACT

The instant disclosure relates to a method of obtaining an extracellular endodextranase enzyme. The method involves inoculating into an aqueous nutrient medium containing dextran and an assimilable nitrogen source, thereby forming a culture, an aerobic bacterial strain which has the following characteristics: GRAM REACTION - POSITIVE SHAPE - RODS MOTILITY - POSITIVE SPORES - SPORULATION DIFFICULT TO DEMONSTRATE IN NUTRIENT AGAR BUT EASIER IN GROWTH LIMITING MEDIUM SUCH AS CITRATE, SPORANGIA DEFINITELY SWOLLEN AGAR COLONIES - ROUND, OPAQUE GLUCOSE AGAR - POSITIVE FOR GROWTH, ACID FROM GLUCOSE USING PEPTONE AS A SOURCE OF NITROGEN GROWTH FACTORS - SLIGHT IF ANY GROWTH IN MINIMAL MEDIA CATALASE - POSITIVE LECITHIN - NO HYDROLYTIC ZONE FORMED MILK AGAR - POSITIVE ZONE OF HYDROLYSIS STARCH - POSITIVE ZONE OF HYDROLYSIS GELATINE AGAR - POSITIVE ZONE OF HYDROLYSIS GLUCOSE ASPARAGINE AGAR - SCANT GROWTH SOYBEAN AGAR - POSITIVE FOR GROWTH NUTRIENT BROTH - UNIFORM TURBIDITY FOLLOWED BY CLEARING AND FORMATION OF SEDIMENT SODIUM CHLORIDE (NaCl) broth - after 2 days incubation, growth in 5% NaCl broth, no growth in 7% NaCl broth NITRITES - NOT PRODUCED FROM NITRATES CITRATE - NOT UTILIZED TYROSINE AGAR - RED-BROWN PIGMENT AFTER 2 DAYS. The culture is then incubated until extracellular endodextranase is elaborated therein. The bacteria are thereafter separated, thereby leaving a solution containing the extracellular endodextranase enzyme. The aerobic bacterial strains used in this method are also disclosed.

waited Mates Patent 1191 Davis et a1.

[ Jan. 22, 1974' [54] METHOD OF PRODUCING DEXTRANASE [75] Inventors: Randall Scott Davis, La Habra; Don

Lee Isenberg, Irvine, both-of Calif.

[22] Filed: Aug. 30, 1971 {21] Appl. No.: 176,314

[52] US. Cl.l 195/65, 195/66 R [51] Int. Cl Cl2d 13/10 [58] Field of Search 195/65, 66 R; 424/50 [56] References Cited UNITED STATES PATENTS 12/1971 'Sipos an 195/66 R 6/1971 Schiff et al 424/50 Primary Examiner-Lionel M. Shapiro Attorney, Agent, or Firm-Robert .l. Steinmeyer; Stanley M. Teigland motility positive spores sporulation difficult to demonstrate in nutrient agar but easier in growth limiting medium such as citrate, sporangia definitely swollen agar colonies round, opaque glucose agar positive for growth, acid from glucose using peptone as a source of nitrogen growth factors slight if any growth in minimal media I catalase positive lecithin no hydrolytic zone formed milk agar positive zone of hydrolysis starch positive zone of hydrolysis v gelatine agar positive zone of hydrolysis glucose asparagine agar scant growth soybean agar positive for growth nutrient broth uniform turbidity followed by clearing and formation of sediment sodium chloride (NaCl) broth after 2 days incubation, growth in 5% NaCl broth, no growth 7 in 7% NaCl broth nitrites not produced from nitrates citrate not utilized tyrosine agar red-brown pigment after 2 days. The culture is then incubated until extracellular endodextranase is elaborated therein. The bacteria are thereafter separated, thereby leaving a solution containing the extracellular endodextranase enzyme. The

aerobic bacterial strains used in this method are also disclosed.

9 Claims, No Drawings 1 METHOD OF PRODUCING DEXTRANASE BACKGROUND OF THE "INVENTION dental plaque, a use presently under thorough investigation.

The above mentioned tasks are preferentiallyv performed using an endodextranase (cleaving glucosidic linkages at random) ratherthan an exodextranase (cleaving glucosidic linkages one at a time from a terminal end). If the enzyme is extracellular, recovery and purification are simplified. In addition, if a culture does not produce. an extracellular dextranase, but rather a cell bound or an intracellular dextranase, the levels of enzyme are usually low. Dextranase production by an aerobic microorganism in short time periods would simplify the fermentation process whereas anaerobic growth is usually slower and more costly.

Molds (Carlson, V.W., andCarlson, W.W., U.S. Pat. No. 2,716,084, Aug. '23, 1955; Carlson, V.W., and Carlson, W.W., U.S. Pat. No. 2,716,237, Aug. 23, 1955; Tsuchuja, H.M.,et al., U.S. Pat. No. 2,742,399, Apr. 17, 1956) have beenused to produce extracellular endodextranase'but the growth period of molds is relatively long making the production costs high. Bacteria have also been found to produce dextranase under certain conditions. An aerobic bacterial strain, a Bacillus sp. (Zevenhuizen, L.P.T.M. 1968. Carbohyd. Res. 6: 310-318) investigated, produced a cell bound exodextranase. Other aerobes, Cytophaga (Janson, J.C., and Porath, J. 1966. Methods Enzymol. 8: 615-621) and Cellvibriofulva (lngelman, B. 1948. Acta Chem. Scand. 2: 803-812) produced endodextranases, but they were v cell bound, and in the case of Cellvibrio the fermentation took several months. Lactobacillus bifidus (Bailey, R.W., and Clarke, R.T.J. 1959. Biochem. J. 72: 49-54; Bailey, R.W., et al. 1960. Nature 186: 553-554; Bailey, R.W., et al' 1961. Biochem. J. 80: 514-519; Bailey, R.W., and Robertson, AM. 1962. Biochem. J. 82: 272-277; Clarke, R.T.J. 1959. J. Gen. Microbiol. 549-553) produces extracellular endodextranase but the organism is anaerobic. Bacteroides (Hehre, E.J., and Sery, T.W. 1952. J. Bacteriol. 63: 424-426; Sery, T.W., and Hehre, E.J. 1956. J. Bacteriol. 71: 373-380) were used to produce both extracellular endoand exodextranases but these o-rganisms also require anaerobic growth conditions.

SUMMARY OF THE INVENTION It is an object of the instant invention to provide an aerobic bacterial strain which has utilization in the production of high levels of extracellular endodextranase in a short time period. The combination of these characteristies advantageously makes the strain useful in an economical method for the production of endodextranase.

The instant invention relates to a method of obtaining an extracellular endodextranase enzyme. The method involves inoculating into an aqueous nutrient medium containing dextran and an assimilable nitrogen source, thereby forming a culture, an aerobic bacterial strain which has the following characteristics:

gram reaction positive shape rods motility positive spores sporulation difficult to demonstrate in nutrient agar but easier in growth limiting medium such as citrate, sporangia definitely swollen agar colonies round, opaque glucose agar positive for growth, acid from glucose using peptone as a source of nitrogen growth factors slight if any growth in minimal media catalase positive lecithin no hydrolytic zone formed milk agar positive zone of hydrolysis starch positive zone of hydrolysis gelatine agar positive zone of hydrolysis glucose asparagine agar scant growth soybean agar positive for growth nutrient broth uniform turbidity followed by clearing and formation of sediment sodium chloride (NaCl) broth after 2 days incubation, growth in 5% NaCl broth, no growth in 7% NaCl broth nitrites not produced from nitrates citrate not utilized tyrosine agar' red-brown pigment after 2 days.

The culture is then incubated until extracellular endodextranase is elaborated therein. The bacteria is thereafter separated, thereby leaving a solution containing the extracellular endodextranase enzyme .which hydrolyzes dextran.

The instant invention also relates to an aerobic bacterial strain which has the following characteristics:

gram reaction positive shape rods motility positive spores sporulation difficult to demonstrate in nutrient agar but easier-in growth limiting medium such as citrate, sporangia definitely swollen agar colonies round, opaque glucose agar positive for growth, acid from glucose using peptone as a source of nitrogen growth factors slight if any growth in minimal media catalase positive lecithin no hydrolytic zone formed milk agar positive zone of hydrolysis starch positive zone of hydrolysis gelatin agar positive zone of hydrolysis glucose asparagine agar scant growth soybean agar positive for growth nutrient broth uniform turbidity followed by clearing and formation of sediment sodium chloride (NaCl) broth after 2 days incubation, growth in 5% NaCl broth, no growth in 7% NaCl broth nitrites not produced from nitrates citrate not utilized tyrosine'agar red-brown pigment after 2 days.

Other objects and advantages of the instant invention will be apparent from the following detailed description.

DETAILED DESCRIPTION The present invention utilizes aerobic bacteria to produce an extracellular endodextranase in short periods of time and at levels much higher than values reported for mold production. Numerous bacteria have been screened for their potential ability to produce dextranase. Over 600 bacterial cultures were isolated that would produce extracellular endodextranase under test cultural conditions. Of these, four organisms were selected which were found to produce high quantities of extracellular endodextranase when so cultivated.

Practically all of the 600 cultures isolated, including the four high producing strains belong to the genus Bacillus. These four microorganisms appear to differ from one another in the amount of extracellular endodextranase produced. These four organisms have been deposited and can be obtained from the permanent collection of the Northern Utilization Research and Development Division of the United States Department of Agriculture, Agricultural Research Service, 1815 North University Street, Peoria, Illinois 61604, with the following deposit numbers: NRRL B-3949, NRRL B- 3976, NRRL B-3977, and NRRL B-3978. The four organisms demonstrate characteristics categorized similarly to those reported for Bacillus coagulans (Breed, R.S., et al., 1957. Bergeys Manual of Determinative Bacteriology. The Williams and Wilkins Company, Baltimore). The characteristics of the four organisms appear in Table 1 below.

TABLE I CHARACTERISTICS OF BACILLUS NRRL 8-3949, NRRL B-3976,

NRRL B-3977, AND NRRL B-3978 Gram reaction: Positive Shape: Rods Motility: Positive Spores: Sporulation difficult to demonstrate in nutrient agar; easier to demonstrate in growth limiting media such as citrate. Under these conditions sporangia definitely swollen.

Agar colonies: Round, opaque Glucose agar: Positive for growth. Acid from glucose using peptone as a source of nitrogen.

Growth factors: Only slight if any growth in minimal media Catalase: Positive Lecithin: No hydrolytic zone formed Milk agar: Positive zone of hydrolysis Starch: Positive zone of hydrolysis Gelatin agar: Positive zone of hydrolysis Glucose asparagine agar: Only scant growth Soybean agar: Positive for growth Nutrient broth: Uniform turbidity followed by clearing and formation of sediment Sodium Chloride (NaCl) broth: After 2 days incubation, growth in by weight NaCl broth but no growth in 7% by weight NaCl broth Nitrites: Not produced from nitrates Citrate: Not utilized Tyrosine agar: Red-brown pigment after 2 days.

According to the instant invention, the bacteria are inoculated into an aqueous medium containing dextran plus an assimilable nitrogen source. If dextran is not incorporated into the medium, extracellular endodextranase production is very low or absent after incubation. Although inorganic sources of nitrogen may be used if supplemented with essential minerals and small amounts of growth factors, higher levels of enzyme are produced when an organic source of nitrogen is used. Corn steep liquor and autolyzed yeast extract were commonly used in the investigations of the present invention. The amounts of dextran and an assimilable nitrogen source can be varied over a wide range and extracellular endodextranase is still produced. However, it was found that the level of enzyme is usually high when dextran and the assimilable nitrogen source are approximately equal in concentration. Under pH controlled conditions it was found that a neutral pH is advantageous for high levels of enzyme production, although this neutral condition was not necessary for more modest levels of enzyme to be produced.

The enzyme produced in these cultures is extracellular, that is, it is present in the cultural medium after the bacteria are removed by centrifugation or filtration. The enzyme has been precipitated by salt or solvent fractionation and can be purified by conventional techniques. t

The following specific examples illustrate the invention. All percentages are weight percentages and all incubations were at a temperature of about 32 C.

EXAMPLES l-l4 Bacillus NRRL B-3949 was examined for its ability to produce dextranase under various cultural conditions. The culture media specified in Table II was sterilized by autoclaving and inoculated with a 5 percent inoculum of the organism. The initial pH of the fermentation was 7.0. After being shaken for the time indicated in the Table, samples were removed and assayed for the presence of extracellular endodextranase. The assay consisted of incubating the enzyme plus dextran and measuring the amount of reducing sugars produced. Assays were performed at pH 6.0 at 40 C. The units used are those defined by Tsuchiya, l-l.M., et al., US. Pat. No. 2,742,399, Apr. 17, 1956.

TABLE II Units/ml Media Organism NRRL B-3949 Ex. Autolyzed 18 Hr. 29 Hr. 41 Hr. No. Yeast Extract Dextran 9 4 2 300 600 600 10 5 2 I20 300 420 ll 3 3 300 600 1200 12 6 3 50 50 50 13 4 4 I80 600 I500 14 5 4 50 l80 200 EXAMPLES l5-28 Bacillus NRRL B-3976 was examined for its ability to produce extracellular endodextranase under various cultural conditions. This culture was grown and its extracellular endodextranase activity assayed as described for Bacillus NRRL B-3949 in the previous examples.

TABLE III Units/ml Media Organism NRRL 13-3976 Ex. Autolyzed l8 Hr. 29 Hr. 41 Hr. No. Yeast Extract Dextran I5 4 50 50 50 16 0.5 0.25 60 150 I80 l7 0.5 0.5 120 330 360 I8 I l 600 900 900 I9 2 l 300 300 300 20 3 1 300 300 300 21 4 1 300 300 480 22 3 2 420 600 600 23 4 2 360 600 900 24 2 I 300 720 25 3 3 420 900 1500 26 6 3 50 50 50 27 4 4 300 900 I500 28 5 4 50 50 50 EXAMPLES 2942 Bacillus NRRL 8-3977 was examined for its ability to produce extracellular endodextranase under various cultural conditions. Growth and assay conditions are identical to those described for Bacillus NRRL B-3949 in Examples 1-14.

TABLE IV Units/ml Media Organism NRRL 8-3977 Ex Autolyzed 18 Br. 29 Hr. 41 Hr. No. Yeast Extract Dextran 29 4 0 50 50 50 30 0.5 0.25 75 I50 180 Ill 0.5 0.5 150 360 360 32 l l 900 I200 I200 33 2 l 240 300 300 34 3 I 240 300 300 35 4 l 180 I80 300 36 3 2 300 600 600 37 4 2 120 300 600 38 5 2 60 I20 480 39 3 3 360 720 900 40 6 3 50 50 50 4| 4 4 120 600 I200 42 5 4 50 I20 I30 EXAMPLES 43-56 Bacillus NRRL 8-3978 was examined for its ability to produce extracellular endodextranase under various cultural conditions. Growth and assay conditions are identical to those described for Bacillus NRRL 13-3949 in Examples l14.

TABLE V Units/ml Media Organism NRRL 8-3978 Ex. Autolyzed [8 Hr. 29 Hr. 4l Hr. No. Yeast Extract Dextran 43 4 0 50 50 50 44 0.5 0.25 60 I I80 45 0.5 0.5 150 300 330 46 l I 600 720 900 47 2 l 300 300 480 48 3 l 300 300 480 49 4 I 300 300 420 50 3 2 360 480 600 51 4 2 300 600 900 52 5 2 I20 600 900 53 3 3 I80 600 900 54 6 3 50 50 50 55 4 4 I80 600 I200 56 5 4 50 I20 It will be appreciated the foregoing description is for illustrative purposes only and the instant invention is not to be limited thereby but only by the claims wherein what is claimed is:

l. The method of obtaining an extracellular endodextranase enzyme which comprises:

a. inoculating into an aqueous nutrient medium containing dextran and an assimilable nitrogen source, thereby forming a culture, an aerobic bacterial strain selected from the group consisting of NRRL B-3949, NRRL B-3976, NRRL B-3977, and NRRL B-3978;

b. incubating the culture untilextracellular endodextranase is elaborated therein; and

c. thereafter separating the bacteria thereby leaving a solution containing the extracellular endodextranase enzyme.

2. The method of claim 1 wherein the bacterial strain is NRRL B-3949.

3. The method of claim 1 wherein the bacterial strain is NRRL B-3976. I

4. The method of claim ll wherein the bacterial strain is NRRL 8-3977.

5. The method of claim 1 wherein the bacterial strain is NRRL B-3978.

6. The method of claim 1 wherein the amount of dextran in the nutrient medium is from about 0.25 percent to about 5 percent by weight.

7. The method of claim 1 wherein the amount of assimilable nitrogen source present is from about 0.5 percent to about 5 percent by weight.

8. The method of claim 1 wherein the assimilable nitrogen source is autolyzed yeast extract.

9. The method of claim 1 wherein the dextran and assirnilable nitrogen source are present in approximately equal concentrations. 

2. The method of claim 1 wherein the bacterial strain is NRRL B-3949.
 3. The method of claim 1 wherein the bacterial strain is NRRL B-3976.
 4. The method of claim 1 wherein the bacterial strain is NRRL B-3977.
 5. The method of claim 1 wherein the bacterial strain is NRRL B-3978.
 6. The method of claim 1 wherein the amount of dextran in the nutrient medium is from about 0.25 percent to about 5 percent by weight.
 7. The method of claim 1 wherein the amount of assimilable nitrogen source present is from about 0.5 percent to about 5 percent by weight.
 8. The method of claim 1 wherein the assimilable nitrogen source is autolyzed yeast extract.
 9. The method of claim 1 wherein the dextran and assimilable nitrogen source are present in approximately equal concentrations. 